Endogenous retrovirus Gag antigen and its gene variants are unique autoantigens expressed in the pancreatic islets of non-obese diabetic mice.

Endogenous retrovirus (ERV) are remnants of ancient retroviruses that have been incorporated into the genome and evidence suggests that they may play a role in the etiology of T1D. We previously identified a murine leukemia retrovirus-like ERV whose Env and Gag antigens are involved in autoimmune responses in non-obese diabetic (NOD) mice. In this study, we show that the Gag antigen is present in the islet stromal cells. Although Gag gene transcripts were present, Gag protein was not detected in diabetes-resistant mice. Cloning and sequencing analysis of individual Gag genes revealed that NOD islets express Gag gene variants with complete open-reading frames (ORFs), in contrast to the diabetes-resistant mice, whose islet Gag gene transcripts are mostly non-ORFs. Importantly, the ORFs obtained from the NOD islets are extremely heterogenous, coding for various mutants that are absence in the genome. We further show that Gag antigens are stimulatory for autoreactive T cells and identified one islet-expressing Gag variant that contains an altered peptide ligand capable of inducing IFN-gamma release by the T cells. The data highlight a unique retrovirus-like factor in the islets of the NOD mouse strain, which may participate in key events triggering autoimmunity and T1D.

Mouse APOBEC3 expression in NIH 3T3 cells mediates hypermutation of AKV murine leukemia virus.

Mouse APOBEC3 (mA3) is a cytidine deaminase that can act on the single-stranded DNA reverse transcripts of retroviruses resulting in G→A hypermutation of proviral DNA. Many mA3 studies have used NIH 3T3 cells assuming that endogenous mA3 production was negligible. We developed a monoclonal antibody specific for mA3 that reveals detectable mA3 in NIH 3T3 cells and we demonstrate that AKV released from the cells undergoes G→A hypermutation. Inactivation of the mA3 gene abolished the deamination confirming that AKV hypermutation was mediated by mA3. The G→A mutations in AKV viral transcripts deviated from a normal distribution with all the mutations contained within only 20% of the transcripts. Single cell analyses revealed that the expression of mA3 in NIH 3T3 cells was limited to 20% of the cells, which likely accounted for the abnormal distribution of mutations. Endogenous NIH 3T3 mA3 was found to restrict AKV replication.

Fatal crashes involving large numbers of vehicles and weather.

INTRODUCTION: Adverse weather has been recognized as a significant threat to traffic safety. However, relationships between fatal crashes involving large numbers of vehicles and weather are rarely studied according to the low occurrence of crashes involving large numbers of vehicles. METHOD: By using all 1,513,792 fatal crashes in the Fatality Analysis Reporting System (FARS) data, 1975-2014, we successfully described these relationships. RESULTS: We found: (a) fatal crashes involving more than 35 vehicles are most likely to occur in snow or fog; (b) fatal crashes in rain are three times as likely to involve 10 or more vehicles as fatal crashes in good weather; (c) fatal crashes in snow [or fog] are 24 times [35 times] as likely to involve 10 or more vehicles as fatal crashes in good weather. If the example had used 20 vehicles, the risk ratios would be 6 for rain, 158 for snow, and 171 for fog. CONCLUSIONS: To reduce the risk of involvement in fatal crashes with large numbers of vehicles, drivers should slow down more than they currently do under adverse weather conditions. Driver deaths per fatal crash increase slowly with increasing numbers of involved vehicles when it is snowing or raining, but more steeply when clear or foggy. PRACTICAL APPLICATIONS: We conclude that in order to reduce risk of involvement in crashes involving large numbers of vehicles, drivers must reduce speed in fog, and in snow or rain, reduce speed by even more than they already do.

Latent murine leukemia virus infection characterized by the release of non-infectious virions.

Clonal cell lines derived from cultures infected with a polytropic MuLV release vastly different levels of infectious virions ranging from undetectable to very high. Low producing clones release an overwhelming proportion of non-infectious virions containing retroviral RNA but deficient in the Env protein. Non-infectious virion production is not due to an inability of the cells to support infectious MuLV production or to an inherent replicative defectiveness of the proviruses. Reinfection of the lowest producing lines with the polytropic or an ecotropic MuLV results in enormous increases in the specific infectivity of the released virions. This indicates a reversible state of retroviral latency characterized by the release of non-infectious virions that is likely the result of insufficient levels of Env protein required for infectivity. The latency state described here may have important roles in in vivo retroviral infections including alterations of the immune response and the production of defective interfering particles.

Endogenous retroviruses mobilized during friend murine leukemia virus infection.

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses.

Analysis of outcomes for single-incision laparoscopic surgery (SILS) right colectomy reveals a minimal learning curve.

BACKGROUND: Single-incision right colectomy has emerged as a safe and feasible alternative to standard laparoscopic resection. As with any new surgical approach, definition of the number of procedures required to optimize the technique is an important goal. Data on this learning curve for single-incision right colectomy are lacking; therefore, we report the outcomes of consecutive single-incision right colectomies to identify the procedural learning curve. METHODS: We retrospectively reviewed consecutive single-incision right colectomies performed by a single surgeon from May 2010 to May 2013. Patients were evaluated in groups of ten to minimize individual patient variability and selection bias. Demographics and peri-operative outcomes among groups were evaluated using ANOVA or Kruskal-Wallis. Statistical improvement was assessed between groups using Student T tests or Mann-Whitney U tests. RESULTS: Seventy consecutive single-incision right colectomies were performed during the study period. There were no differences in patient demographics over the course of the experiences, suggesting that the selection bias did not influence the outcomes. There was a statistical improvement in operative time after the first 10 cases (103 vs. 130 min, p = 0.01). A second statistical improvement in operative time occurred after 40 cases (97 vs. 114 min, p = 0.03). There was no statistical improvement in estimated blood loss, lymph node harvest, conversion rate, length of stay, or post-operative morbidity throughout the experience. CONCLUSIONS: Analysis of our large series of consecutive cases indicates that for a surgeon trained in advanced laparoscopic techniques and given adequate case volume, the outcomes from the procedure are quickly optimized with a minimal learning curve. Operative time is optimized following 40 procedures. Identification of the learning curve is critical for surgeons wishing to implement a single-incision approach and to ensure that the outcomes are optimized prior to thorough comparison with standard laparoscopic or open approaches.

Chinese traffic fatalities and injuries in police reports, hospital records, and in-depth records from one city.

OBJECTIVES: Claims of sharp reductions in Chinese traffic casualties after 2002 based on police-reported data have been questioned in the literature. The objective of this study is to determine whether a decline in casualties occurred and to better understand the police data. METHODS: The first of 2 unrelated studies analyzed data from 210 military hospitals throughout China providing records for inpatients injured in traffic accidents (2001-2007). The second compared in-depth crash records (2000-2006) from one city to officially released data. RESULTS: Hospital data showed that casualties increased from 2002 to 2007. The city investigation showed consistently far more fatalities and injuries in the in-depth data than officially released. For example, in-depth data showed 1,720 fatalities. Only 557 of these were reported officially (data loss = 68%). Disaggregating into 3 regions showed a data loss of 41% in urban areas, 63% in rural areas, and 90% in rural-urban fringe zones. For injuries, data losses were even greater. CONCLUSIONS: Traffic fatalities and injuries did not decrease from 2002 to 2006. The in-depth city data contained 3 times as many fatalities and 5 times as many injuries as reported by police. Reasons why this occurred and suggestions to improve data collection and reduce casualties are given.

Traffic fatality reductions: United States compared with 25 other countries.

OBJECTIVES: I compared US traffic fatality trends with those in 25 other countries. METHODS: I have introduced a new measure for comparing safety in different countries: traffic deaths in a specific year relative to largest annual number recorded. I used only data from the International Road Traffic Accident Database. RESULTS: The United States is a unique outlier. Fatalities in all 25 other countries declined further after reaching their maximum values. For example, the United States and the Netherlands both reached maximum values in 1972. From 1972 to 2011 US deaths declined by 41%, whereas those in the Netherlands declined by 81%. If US fatalities had declined by 81% there would have been 22 000 fewer US road deaths in 2011. If the United States matched percentage declines of 6 additional countries, US deaths would have declined by more than 20 000. CONCLUSIONS: If US traffic deaths had declined by the same percentage as in any 1 of 7 other countries, more than 20 000 fewer Americans would have been killed in 2011.

Incorporation of mouse APOBEC3 into murine leukemia virus virions decreases the activity and fidelity of reverse transcriptase.

APOBEC3 proteins are restriction factors that induce G→A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G→A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.

Analysis of two monoclonal antibodies reactive with envelope proteins of murine retroviruses: one pan specific antibody and one specific for Moloney leukemia virus.

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.

Three Sgp loci act independently as well as synergistically to elevate the expression of specific endogenous retroviruses implicated in murine lupus.

Endogenous retroviruses are implicated in murine lupus nephritis. They provide a source of nephritogenic retroviral gp70-anti-gp70 immune complexes through the production of serum gp70 protein and anti-gp70 autoantibodies as a result of the activation of TLR7. The Sgp (serum gp70 production) loci identified in lupus-prone mice play distinct roles for the expression of different classes of endogenous retroviruses, as Sgp3 regulates the transcription of xenotropic, polytropic and modified polytropic (mPT) viruses, and Sgp4 the transcription of only xenotropic viruses. In the present study, we extended these analyses to a third locus, Sgp5, using BALB/c mice congenic for the NZW-derived Sgp5 allele and also explored the possible interaction of Sgp3 and Sgp4 loci to promote the expression of endogenous retroviruses and serum gp70. The analysis of Sgp5 BALB/c congenic mice demonstrated that the Sgp5 locus enhanced the expression of xenotropic and mPT viruses, thereby upregulating the production of serum gp70. These data indicate a distinct action of the Sgp5 locus on the expression of endogenous retroviruses, as compared with two other Sgp loci. Moreover, comparative analysis of C57BL/6 double congenic mice for Sgp3 and Sgp4 loci with single congenic mice revealed that Sgp3 and Sgp4 acted synergistically to elevate the transcription of the potentially replication-competent Xmv18 provirus and the production of serum gp70. This indicates that the combined effect of three different Sgp loci markedly enhance the expression of endogenous retroviruses and their gene product, serum gp70, thereby contributing to the formation of nephritogenic gp70-anti-gp70 immune complexes in murine lupus.

Single-incision laparoscopic splenectomy: preliminary experience in consecutive patients and comparison to standard laparoscopic splenectomy.

BACKGROUND: Since first being described in 2009, single-incision laparoscopic splenectomy has been described in a limited number of case reports and small case series. No studies have evaluated single-incision splenectomy in unselected patients, and outcomes of the procedure have not previously been compared to standard laparoscopy. METHODS: A retrospective review was conducted to evaluate all single-incision splenectomies performed by a single surgeon between June 2010 and June 2011. Additionally, patients who underwent standard laparoscopic splenectomy by surgeons in the same tertiary referral surgical oncology group were evaluated to serve as a control group. Demographic data, operative parameters, and postoperative outcomes were assessed. RESULTS: Eight patients underwent successful single-incision splenectomy during the study period without conversion to an open procedure or requiring additional ports. The median operative time was 92.5 min. There was 25 % morbidity and no mortality in the study group. Median length of stay was 4 days. Additionally, 18 patients who underwent standard laparoscopic splenectomy were evaluated for comparison. No significant differences were identified in the preoperative patient characteristics between the two groups. Single-incision splenectomy was associated with a shorter operative time (92.5 vs. 172 min, p = 0.003), lower conversion rate, equivalent length of stay, reduced mortality, similar morbidity, and comparable postoperative narcotic requirements. CONCLUSIONS: Single-incision splenectomy is feasible, safe, and efficient in an unselected patient population in the hands of an experienced laparoscopic surgeon. The single-incision technique is comparable to standard laparoscopic splenectomy in terms of operative time and perioperative outcomes.

Sgp3 and TLR7 stimulation differentially alter the expression profile of modified polytropic retroviruses implicated in murine systemic lupus.

The envelope glycoprotein, gp70, of endogenous retroviruses represents one of the major nephritogenic autoantigens implicated in murine systemic lupus erythematosus. Among different endogenous retroviruses (ecotropic, xenotropic and polytropic), lupus-prone mice express remarkably high levels of modified polytropic (mPT) retroviruses, which are controlled by the Sgp3 (serum gp70 production) locus. To define the contribution of the Sgp3 locus derived from lupus-prone mice to the expression of the specific mPT proviruses, the genetic origin of different mPT viruses expressed in livers and thymi of wild-type and Sgp3 congenic C57BL/6 mice was determined through clonal analysis of their transcripts. Among 13 mPT proviruses present in the C57BL/6 genome, only 3 proviruses (Mpmv6, Mpmv10 and Mpmv13) were selectively but differentially expressed in livers and thymi. This was likely a result of co-regulated expression with host genes because of their integration in the same transcriptional direction. In contrast, Sgp3 induced the steady-state expression of an additional select group of mPT proviruses and, after stimulation of TLR7, the highly upregulated expression of a potentially replication-competent mPT virus Mpmv4. These results indicated that the expression of distinct subpopulations of mPT retroviruses was regulated by Sgp3- and TLR7-dependent mechanisms. The induction of potentially replication-competent mPT viruses and the upregulation of one such virus after stimulation with TLR7 in Sgp3 congenic mice further highlight the implication of Sgp3 in autoimmune responses against nephritogenic serum gp70 through the activation of TLR7.

Profound amplification of pathogenic murine polytropic retrovirus release from coinfected cells.

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.

Single-incision laparoscopic right colectomy in an unselected patient population.

BACKGROUND: Single-incision laparoscopic surgery has become increasingly utilized for colorectal surgery, with the most frequently reported single-incision laparoscopic operation being right hemicolectomy. While current data suggest that single-incision laparoscopic right colectomy is feasible, safe, and roughly equivalent to standard laparoscopic right colectomy, the technique has to date only been described in highly selected patients. Single-incision laparoscopic right colectomy has not yet been assessed in a standard patient population. METHODS: A retrospective review was conducted to evaluate all single-incision right hemicolectomies performed by a single surgeon between May 2010 and April 2011. Demographic data, operative parameters, and postoperative outcomes were assessed. RESULTS: Single-incision laparoscopic colectomy was performed in a series of 30 consecutive patients with indications for right colectomy. One patient required conversion to an open procedure for extensive adhesions, while no patients required additional port placement. Mean operative time was 107 min. All patients had negative margins and had an average of 20 lymph nodes harvested. Mean length of stay was 6 days. There were no intraoperative complications and no mortality in the study. The perioperative complication rate was 37%, with 71% of complications being grade 1. CONCLUSIONS: Single-incision laparoscopic colectomy is feasible, safe, efficient, and oncologically sound for most patients who are seen in a typical colorectal practice. These data are useful as single-incision laparoscopic colectomy becomes more broadly implemented.

Sgp3 and Sgp4 control expression of distinct and restricted sets of xenotropic retroviruses encoding serum gp70 implicated in murine lupus nephritis.

The envelope glycoprotein gp70 of endogenous retroviruses implicated in murine lupus nephritis is secreted by hepatocytes and its expression is controlled by Sgp3 (serum gp70 production 3) and Sgp4 loci derived from lupus-prone mice. Among three different endogenous retroviruses (ecotropic, xenotropic and polytropic), xenotropic viruses are considered to be the major source of serum gp70. Although the abundance of xenotropic viral gp70 RNA in livers was up-regulated by the presence of these two Sgp loci, it has not yet been clear whether Sgp3 and Sgp4 regulate the expression of a fraction or multiple xenotropic viruses present in mouse genome. To address this question, we determined the genetic origin of xenotropic viral sequences expressed in wild-type and two different Sgp congenic C57BL/6 mice. Among 14 xenotropic proviruses present in the C57BL/6 genome, only two proviruses (Xmv10 and Xmv14) were actively transcribed in wild-type C57BL/6 mice. In contrast, Sgp3 enhanced the transcription of Xmv10 and induced the transcription of three additional xenotropic viruses (Xmv15, Xmv17 and Xmv18), while Sgp4 induced the expression of a different xenotropic virus (Xmv13). Notably, stimulation of TLR7 in Sgp3 congenic C57BL/6 mice led to a highly enhanced expression of potentially replication-competent Xmv18. These results indicated that Sgp3 and Sgp4 independently regulated the transcription of distinct and restricted sets of xenotropic viruses in trans, thereby promoting the production of nephritogenic gp70 autoantigens. Furthermore, the induced expression of potentially replication-competent xenotropic viruses by Sgp3 may contribute to the development of autoimmune responses against gp70 through the activation of TLR7.

Noninfectious retrovirus particles drive the APOBEC3/Rfv3 dependent neutralizing antibody response.

Members of the APOBEC3 family of deoxycytidine deaminases counteract a broad range of retroviruses in vitro through an indirect mechanism that requires virion incorporation and inhibition of reverse transcription and/or hypermutation of minus strand transcripts in the next target cell. The selective advantage to the host of this indirect restriction mechanism remains unclear, but valuable insights may be gained by studying APOBEC3 function in vivo. Apobec3 was previously shown to encode Rfv3, a classical resistance gene that controls the recovery of mice from pathogenic Friend retrovirus (FV) infection by promoting a more potent neutralizing antibody (NAb) response. The underlying mechanism does not involve a direct effect of Apobec3 on B cell function. Here we show that while Apobec3 decreased titers of infectious virus during acute FV infection, plasma viral RNA loads were maintained, indicating substantial release of noninfectious particles in vivo. The lack of plasma virion infectivity was associated with a significant post-entry block during early reverse transcription rather than G-to-A hypermutation. The Apobec3-dependent NAb response correlated with IgG binding titers against native, but not detergent-lysed virions. These findings indicate that innate Apobec3 restriction promotes NAb responses by maintaining high concentrations of virions with native B cell epitopes, but in the context of low virion infectivity. Finally, Apobec3 restriction was found to be saturable in vivo, since increasing FV inoculum doses resulted in decreased Apobec3 inhibition. By analogy, maximizing the release of noninfectious particles by modulating APOBEC3 expression may improve humoral immunity against pathogenic human retroviral infections.